Uporedna analiza morfoloških promena i distribucije virusnog antigena u organima labudova i kokošaka prirodno inficiranih virusom avijarne influence podtipa H5N8

Abstract

Highly pathogenic avian influenza viruses cause infections in birds, a large number of domestic and wild animals as well as in humans, and are the cause of serious veterinary and public health concern. The distinct genetic variability and instability of the influenza virus genome enable the creation of a large number of different subtypes of avian influenza virus with altered characteristics in terms of antigenic properties, pathogenicity and host specificity. In 2014, a novel reassortant subtype of highly pathogenic avian influenza H5N8 was detected for the first time on the European continent. In the Republic of Serbia, the first outbreak of avian influenza subtype H5N8 was recorded at the end of 2016. The highest mortality rate during this outbreak was detected in the mute swans (Cygnus Olor) and the virus was also diagnosed in a small number of domestic hens in rural households. The aim of this PhD thesis was a comparative analysis of macroscopic and microscopic lesions, distribution and virus tropism as well as the determination of the inflammatory cell immunophenotyping in the organs of two different bird species naturally infected with the avian influenza virus subtype H5N8. The carcasses of 15 mute swans and 15 hens that died during the avian influenza outbreak were examined. In addition, the aim of this research was the identification and molecular characterization of the avian influenza virus detected in Serbia during the winter 2016-2017. The real-time RT-PCR method was performed for detection of H5N8 HPAI virus presence in materials originating from naturally infected birds. Pathohistological and immunohistochemical methods were used to determine the character of lesions, the distribution of viral antigen and immunophenotyping of inflammatory cells in the lung, heart, spleen, intestine, kidney, pancreas and brain tissue. Selected tissue samples were macroscopically analyzed, fixed in formalin and embedded into paraffin blocks. Tissue sections were stained with hematoxylin-eosin and an immunohistochemically using the Novolink Polymer Detection System commercial kit. Antibodies for avian influenza nucleoprotein, CD3, CD21 and GFAP were used as primary antibodies. A semi-quantitative analysis was used for estimating the distribution of viral antigen in different tissues. The presence of the highly pathogenic avian influenza virus H5N8 was confirmed in all tested bird samples using the molecular-genetic method (real-time RT-PCR). Phylogenetic analysis performed using MEGA 6.0 software found that the virus belongs to the cluster 2.3.4.4. group B influenza viruses that circulated in the area of the European continent during 2016 and 2017. The detected virus isolate is related to isolates found in Hungary, Croatia and Macedonia. Macroscopic examination of organ samples from mute swans revealed the most frequent changes were haemorrhages, necrosis and congestion in heart, pancreas, spleen and lungs. The most frequent pathomorphologic changes observed in hens were haemorrhages of the lungs and heart as well as the presence of necrosis of the spleen. A very significant difference was observed in the incidence of necrosis and haemorrhages of the pancreas in hens and swans. This finding was constant in all infected swans, while in hens were no macroscopic lesions in this organ. In addition, a significant difference was observed in the macroscopic finding of the kidneys, where haemorrhages were observed in the hens, while there were no macroscopic lesions in the kidneys of mute swans. Microscopic examination of the organs of both bird species revealed necrosis and haemorrhages of varying intensity, lymphocyte infiltrate accumulation, and hyperemia of the blood vessels in most organs. Character and severity of microscopic lesions were similar in both bird species. The only significant difference was the presence of haemorrhages in the pancreas of mute swans, while this finding wasn’t observed in the pancreas of the hens.The viral antigen was detected in all examined organs using the immunohistochemical method, except in the intestines of mute swans. This finding may indicate that in this bird species the primary route of virus dissemination is through the respiratory tract. On the other hand, the viral antigen is abundantly expressed in the lungs and intestinal epithelium of the hens, indicating that excretion of the virus occurs through the intestinal and respiratory system in this bird species. Differences in expression of viral antigen are found in the heart, lungs and intestines. In these organs, the viral antigen is significantly more frequent and more detectable in chickens than in the swans. The most abundant expression of viral antigen in both bird species was observed in the brain and pancreas. The immunohistochemical method has demonstrated that avian influenza virus H5N8 had a tropism to the monocyte-macrophage cells, epithelial, endothelial, nerve cells, including epithelial cells of the renal tubules, enterocytes and bronchoalveolar epithelium. The appearance of nervous symptoms of the disease, large amounts of viral antigen in the brain, and large amounts of GFAP + astrocyte in both bird species indicate the neurotrophic characteristic of the virus. In order to determine the phenotypic profile of inflammatory cell infiltrate in organs of infected birds, immunohistochemistry was applied. The dominant cell population in the inflammatory infiltrate was CD3+ cells (T lymphocytes), while CD21+ (B lymphocytes) were present in a very small number.